This invention relates to the production and use of polarization labels for identifying three dimensional orientation, more specifically, three dimensional orientation of light emitting and absorbing objects.
Fluorescent labeling is a very powerful technique for locating and tracking tagged objects, with applications in areas ranging from molecular dynamics and combinatorial chemistry to security systems and inventory control. However, this technique has several limitations. The number of objects which can be tracked simultaneously is limited by the fluorescent wavelengths available from the tags. In addition, this technique cannot provide information about three dimensional orientation relationships among objects.
A complete description of a three dimensional (3D) system includes analysis of the orientation of its components with respect to each other. For example, the chemical interactions of proteins with each other depend intimately on the spatial relationships they have with each other in all three dimensions. Characterization of an interface requires not only knowledge of the chemistry of the materials at either side of that interface but also of their orientation with respect to the interface and to each other.
Some 3D information about individual molecules or other complexes is available through scanning near-field optical microscopy (SNOM). However, SNOM is a relatively invasive technique which, in addition, can only analyze one small region of a sample at a time. Thus, the technique is not useful for high-throughput screening. Because of the small field of view, only a small total area can be analyzed, resulting in sampling errors. As a result, if a sample exhibits large amounts of variation, SNOM may not reveal the full variability of the sample. While far field microscopy can probe larger areas, it can only provide two-dimensional information about the orientation of a molecule with respect to the plane of the sample under observation.
In one aspect, the method of determining the orientation of a photoactive moiety exhibiting an anisotropic transition dipole includes exposing the moiety to a light source to stimulate a spectral emission for the moiety and correlating the emission with the orientation of the moiety. The moiety exhibits spectral emission polarized along at most two dimensions. In one embodiment the moiety exhibits emission polarized along two dimensions. The moiety may include a particle from the group consisting of a crystalline arrangement of photoactive molecules and a photoactive nanocrystal. The moiety may include a matrix in which photoactive objects exhibiting an anisotropic emission dipole are embedded. The matrix may be a polymer. In another embodiment, the photoactive objects include a member of the group consisting of a photoactive molecule, a single crystal of photoactive molecules, and a photoactive nanocrystal. The photoactive moiety may further include a magnetic or polar moiety. The photoactive moiety may be photobleached to produce the anisotropy. The transition dipoles of the photoactive objects can be aligned with the photoactive moiety.
In another aspect, a photoactive moiety according to the invention exhibits an anisotropic transition dipole wherein the moiety exhibits emission of polarized light in response to energy absorption. The moiety may include a particle from the group consisting of a crystalline arrangement of photoactive molecules and a photoactive nanocrystal. In one embodiment, the moiety includes a matrix in which photoactive objects exhibiting an anisotropic emission dipole are embedded. In this embodiment, the photoactive objects include a member of the group consisting of a photoactive molecule, a single crystal of photoactive molecules, and a photoactive nanocrystal. The transition dipoles of the photoactive objects may be aligned within the photoactive moiety. The moiety may be photobleached to produce the anisotropy. In one embodiment, the photoactive objects have a one-dimensional transition dipole in their natural state and the moiety emits light polarized in two dimensions. The moiety may emit light polarized in one dimension. In another embodiment, the photoactive objects have a two-dimensional transition dipole in their natural state and the moiety exhibits emission of light polarized in two dimensions.
In yet another aspect, the method of the invention for creating a moiety of photoactive molecules includes entrapping the photoactive molecules in a solid, the photoactive molecules exhibiting an oriented transition dipole, and photobleaching a portion of the photoactive molecules. The moiety will then exhibit polarized light emission in response to light absorption. In one embodiment of this aspect of this invention, the solid is a matrix in which the photoactive molecules are entrapped and the matrix includes a polymer. In this embodiment, the photoactive molecules have a uni-dimensional transition dipole in their natural state. In this invention, the moiety is photobleached with polarized light and after photobleaching the moiety emits light polarized in two dimensions.
In yet another aspect, the method of the invention for providing an identification unit includes selecting an item of interest and providing an identifier wherein the identifier includes at least one particle having characteristic spectral emission. One or more reactive moieties are provided which are attached to the surface of the particle wherein the one or more reactive moieties are selected for their ability to be compatible with the item of interest. The spectral emission of the at least one particle is at least characterized by polarization. In one embodiment, the item of interest is selected from the group consisting of identification tag, security tag, consumer product, fluid, gas, solid, biomolecule, and chemical compound. In another embodiment, the spectral emission of the at least one particle is further characterized by wavelength, intensity, or both.
In yet another aspect, the invention is a library of items of interest wherein each item of interest has associated with it one or more identifiers wherein the one or more identifiers each comprise a particle with a characteristic spectral emission in which the spectral emission is characterized at least in part by polarization. In one embodiment of this aspect of the invention, the item of interest is selected from the group consisting of identification tag, security tag, consumer product, fluid, gas, solid, biomolecule, and chemical compound. The spectral emission of the at least one particle is further characterized by wavelength intensity or both.
Another aspect of the invention is a method of tracking the motion of an item of interest and includes providing an item of interest, wherein the item of interest has associated with it at least one particle having a characteristic spectral emission wherein the spectral emission of the at least one particle is characterized at least in part by polarization. The at least one particle is exposed to an energy source to stimulate the spectral emission and the spectral emission is correlated with the item of interest. These steps are repeated at known intervals.
Still another aspect of the invention is a method of tracking the change in orientation of an item of interest while the item is in motion. The method includes providing an item of interest, wherein the item of interest has associated with it at least one particle having a characteristic spectral emission wherein the spectral emission of at least one particle is characterized at least in part by polarization in two dimensions. The at least one particle is exposed to an energy source to stimulate the spectral emission and the spectral emission is correlated with the orientation of the item of interest. These steps are repeated at known intervals.
In yet another aspect, the invention is method of tracking the change in conformation of an item of interest including providing an item of interest wherein the item of interest has associated with it a plurality of photoactive particles having characteristic spectral emissions and exposing the particles to an energy source to stimulate the spectral emissions. The spectral emissions are correlated with the conformation of the item of interest. Thereafter, these steps are repeated at known intervals. The spectral emissions are characterized at least in part by polarization. In another aspect, the invention describes a method of tracking fluid flow. Identifiers according to the invention are exposed to a light source, such that they will exhibit emission of light in one dimension. The emission is correlated with the position and orientation of at least a portion of the identifiers. The method is repeated to track the change in position (motion). In one embodiment, the motion is tracked sequentially within a series of focal planes. That is, the motion is tracked in one focal plane, and then the focal length adjusted and the motion tracked again.
In another aspect, the invention describes an apparatus for detecting the orientation of a photoactive particle or other item of interest to which the particle might be attached. A detector according to the invention comprises at least three beam splitting mirrors, each of which has a polarizing filter associated with it, and at least one photon detector. The detector also includes means to correlate the spectral emission of the particle to its location and the orientation of its emission, which may include a confocal lensing system, a monochromator, or light filters. The detector may further comprise a second plurality of beam splitting mirrors associated with each polarizing filter and light filters associated therewith.